Erythritol-producing moniliella strains

ABSTRACT

An isolated strain of the Moniliella species that converts glucose to erythritol with a conversion rate of at least about 45% is disclosed, as is a method of producing erythritol from such a strain.

BACKGROUND

[0001] Erythritol is a sugar alcohol that can be found in lichens, hempleaves, and mushrooms. It is also savored in fermented foods such aswine, soya sauce, or saki (Sasaki, T. (1989) Production technology oferythritol. Nippon Nogeikagaku Kaishi 63: 1130-1132). Erythritol is afour-carbon polyol, which possesses several properties such as sweetness(about 70-80% of sucrose), tooth friendliness, very low calorific value(0.3 kcal/g, a tenth of sucrose), non-carcinogenicity and, unlike otherpolyols, causes little, if any, gastro-intestinal discomfort (Harald andBruxelles (1993) Starch/Starke 45:400-405).

[0002] Traditional industrial erythritol production is carried out byadding catalysts such as hydrogen and nickel to the raw material sugarsunder the environment of high temperature and high pressure. Anotherprocess is performed by the chemo-reduction of raw materials such asmeso-tartarate (Kent, P. W., and Wood, K. R. (1964) J. Chem. Soc.2493-2497) or erythrose (Otey, F. H., and Sloan, J. W. (1961) Ind. Eng.Chem. 53:267) to obtain erythritol. In addition, erythritol can beproduced by a number of microorganisms. Such organisms include highosmophilic yeasts, e.g., Pichia, Candida, Torulopsis, Trigonopsis,Moniliella, Aureobasidium, and Trichosporon sp. (Onishi, H. (1967) HakkoKyokaish 25:495-506; Hajny et al. (1964) Appl. Microbiol. 12:240-246;Hattor, K., and Suziki, T. (1974) Agric. Biol. Chem. 38:1203-1208;Ishizuka, H., et al. (1989) J. Ferment. Bioeng. 68:310-314.)

SUMMARY

[0003] The invention features isolated strains of the Moniliella specieswith enhanced capacities for the conversion of glucose to erythritol.Such strains can produce erythritol from glucose with a conversion rateof at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or greater underoptimal conditions.

[0004] Strains of the invention include isolates of Moniliella from anatural source; and the mutants of a Moniliella strains, e.g., aMoniliella strains assigned the American Type Culture Collection (ATCC)accession numbers of PTA-1227, PTA-1228, PTA-1229, PTA-1230, andPTA-1232. One particular mutant strain is the isolated strain,N61188-12, deposited with the American Type Culture Collection with theaccession number ______.

[0005] As used herein, the term “mutant” refers to a strain whosegenetic composition differs by at least one nucleotide, e.g., asubstitution, insertion, or deletion, relative to a reference or parentstrain. A mutant of the invention can be produced by a number ofmethods. One method is the selection of strains with increasederythritol conversion rates relative to a parent strain. The strains canbe obtained by random mutagenesis of the parent strain, e.g., by meansof a chemical mutagen, a transposon, or irradiation. In addition, amutant strain of the invention can include a recombinant nucleic acidsequence. For example, a mutant may be a strain that harbors anadditional nucleic acid sequence, e.g., a sequence transformed,transduced, or otherwise inserted into a cell of the parent strain. Theadditional nucleic acid sequence can encode a polypeptide that isgenerally or conditionally expressed. Alternatively, the additionalnucleic acid sequence can encode a nucleic acid sequence capable ofaltering cell physiology, e.g., an anti-sense, a ribozyme, or othernucleic acid sequence. In another instance, the inserted nucleic acid isinserted into an endogenous gene, and alters (e.g., enhances ordisrupts) its function. For example, the inserted nucleic acid can be aknockout construct that inactivates the endogenous gene; or anartificial enhancer or promoter that increases transcription of theendogenous gene. The mutation can disrupt the ability of the parentalstrain to import, assimilate, or consume erythritol or mannitol.

[0006] The invention also features a method of producing erythritol. Themethod includes growing a Moniliella strain of the invention, e.g., anenhanced mutant, in a culture; and purifying erythritol from theculture, e.g., from the supernatant or from the cell pellet.

[0007] The details of one or more embodiments of the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

DETAILED DESCRIPTION

[0008] The fungus Moniliella is capable of fermenting simple sugars toproduce erythritol, a well-relished component of many cuisines.Screening and mutagenesis are used to identify improved strains ofMoniliella that are capable of highly efficient erythritol productionyields. Such strains are ideal for large-scale erythritol production, ascan be achieved by the exemplary methods described herein.

[0009] Isolation of Enhanced Erythritol Producing Strains

[0010] Isolates of Moniliella can be obtained from a natural source asdescribed in U.S. patent application Ser. No. 09/585,926, filed Jun. 2,2000. For example, isolates of Moniliella can be obtained naturalsources having high sugar content include honey, preserved fruit, andpollen. Each strain is identified based on its capability to convertglucose to erythritol and its various morphological and physiologicaltraits. As used herein, the “glucose-to-erythritol conversion rate” isdefined as the amount of erythritol produced divided by the amount ofglucose consumed. The resulting ratio can be expressed as a percentage.The glucose-to-erythritol conversion rate of a fungal strain can becalculated by the following method. The strain is first cultured in a10-ml broth containing 30% glucose and 1% yeast extract (initial celldensity 1·10⁵ cells/ml) in a 50 ml flask in a rotary shaker at 150 rpmand 30° C. for 6 days. Then, both the concentration of erythritol in themedium and the concentration of glucose in the medium are determined.The conversion of 1 g of glucose into 0.3 g of erythritol is termed a30% conversion rate. The morphological traits are determined followinggrowth on 4% malt extract, 0.5% yeast extract agar for 10 days at 20° C.See The Yeasts, A Taxonomic Study, Edited by Kurtzman et al., 4th Ed.,page 785, Elsevier, Amsterdam (1998).

[0011] A mutant of a Moniliella strain can be obtained by themutagenesis method described in Ishizuka, et al. (1989) J. Ferment.Bioeng. 68:310-314, or a variation thereof (see also U.S. Pat. No.5,036,011). One variation for the mutagenesis of Moniliella cells withN-methyl-N-nitrosoguanidine (NTG) is described as follows. Moniliellacells are inoculated in broth with 30% glucose and 1% yeast extract, andcultured overnight at 30° C. on a rotary shaker at 150 rpm. This cultureis diluted 1:100 into 10 ml of broth with 30% glucose, and incubated at30° C. on a rotary shaker at 150 rpm for 1 day. The culture broth iscentrifuged at 3,000 rpm for 15 min to form a cell pellet and thesupernatant is discarded. The cell pellet is washed with 10 ml ofsterile 0.1 M pH 7.0 phosphate buffered saline (PBS). The suspension iscentrifuged (3,000 rpm, 15 min) and the supernatant is again discarded.The cells are resuspended in PBS, with 150 μg/ml NTG for 10 minutes.

[0012] After treatment with NTG, the Moniliella cells are grown in aglucose solution for 3 hours. The culture is then diluted appropriatelyand spread onto the medium containing 65% glucose and incubated at 30°C. for 6 days. Colonies are selected randomly, inoculated into brothcontaining 30% glucose, and incubated at 30° C. on a rotary shaker at150 rpm overnight. A 1:100 dilution of the overnight culture is used toinoculate into a 30% glucose solution (10 ml) that is incubated at 30°C. on a rotary shaker at 150 rpm for 4 days. The medium from thisculture is then centrifuged at 12,000 rpm for 10 min. The supernatant isdiluted appropriately and the amount of residual glucose is measuredusing the DNS method (see below). Cultures with higher glucoseconsumption (i.e., lower residual glucose) are further analyzed todetermine erythritol yield. The HPLC method described below can be usedto quantitate erythritol yield. Cultures with indications of elevatederythritol yield are subject to further verification. For example,individual colonies are obtained for the culture, re-grown as describedabove, and reanalyzed. Selected colonies can be improved by additionalrounds of mutagenesis according to these procedures.

[0013] Measurement of Residual Glucose

[0014] 4-day-old culture broth is collected and centrifuged at 12,000rpm for 10 min. The supernatant is diluted appropriately. 1 ml of eachdiluted solution is added to 0.5 ml of DNS (dinitrosalicylic acid)reagent. DNS reagents (e.g., a. 1% 3,5-dinitrosalicylic acid (DNS). b.0.2% phenol; c. 0.05% NaHSO₃ or 0.025% Na₂S₂O₃; d. 1% NaOH; e. 0.5%potassium sodium tartrate tetrahydrate) were prepared and used accordingto method described in Miller, G. L. (1958) Anal. Chem. 31:426-428. Themixture is mixed well and incubated at 100° C. for 5 min. After coolingunder room temperature, 9 ml water is added and the absorbance at 540 nm(OD₅₄₀ nm) is determined. The absorbance at 540 nm is used to determinethe concentration of glucose by comparison with the standard curve,obtained by measuring pure glucose at various concentrations.

[0015] Measurement of Erythritol Concentration

[0016] The amount of erythritol in a supernatant can be quantitated byHPLC and TLC, e.g., to determine the erythritol-producing capacity of astrain. HPLC analysis is performed by Hewlett Packard H4033A analyzer onan Ion-300 chromatography column, using 0.1 N sulfuric acid as theflowing phase with a flowing rate of 0.4 ml/min, the temperature beingset at 75° C. For TLC analysis, the Neissner et al. procedure isfollowed. (Neissner, et al. 1980. Herstellung, aanalyse und DC-trennungvon fettsaure erythritpartialestem. FETTE SEIFEN ANSTRICHMITTEL.82:10-16.). After rinsing Kieselgel 60F254 (Merck) with 4% boric acid,the gel is heated in an incubator at 105° C. for 20 minutes before use.The spreading solvent is ethylmethylketone:acetone:water (100:10:10 byvol.) and the color-developing agent is KMnO₄ in concentrated sulfuricacid.

[0017] Erythritol purified from a supernatant by HPLC or TLC can befurther purified by extraction and then dried under reduced pressure.The further purified product and an erythritol standard are acetylatedaccording to the method of Shindou et al. (Shindou et al. 1989. J.Agric. Food Chem. 37:1474-1476.). Erythritol standards are commerciallyavailable, e.g., from Merck, Germany. The resulting sample can beassayed by GC-MS to determine if the re-purified product was identicalto that of the standard sample.

[0018] Large Scale Production of Erythritol

[0019] Following the specific examples provided below, a skilled artisancan optimize erythritol yield of a mutant Moniliella strain byidentifying preferred pH, temperature, and carbon source for growth andfermentation. Similar analysis can be used to optimize aeration,stirring speed, culture volume, and culture time.

[0020] To produce erythritol on a larger scale, 0.2 ml of Moniliellacells preserved in glycerol are added to 50 ml of broth in a 500 mlflask, and incubated at 30° C. on a rotary shaker at 150 rpm for about24 hours. From this culture, 2 ml are used to inoculate a second 500 mlflask with 50 ml of broth. The second culture is incubated at 30° C. ona rotary shaker at 150 rpm for 48 hours. The second culture broth isused to inoculate 2 L of broth in a 5 L fermentor (NBS. Edison, N.J.,USA). The culture conditions are as follows. Aeration: 1 VVM; stirredspeed: 500 rpm; temperature: 30° C.; culture period: 5-7 days.

[0021] For these purposes, the broth can consist of 30%, 35%, 40%, 45%,or 50% glucose, together and 1% yeast extract. In addition, KM72 andKM72F (Shin Etsu, Shin-Etsu Chemical Co., Ltd. 6-1, Ohtemachi 2-chome,Chiyoda-ku, Tokyo, Japan) can be used as a defoamer.

[0022] Purification of Erythritol

[0023] Media from the fermentor is centrifuged to separate the culturesupernatant from pelleted cells. The supernatant is decolored by passageover active carbon (e.g., powdered carbon as can be obtained from alocal supplier). The decolored supernatant is desalted andde-proteinated by consecutive passage of over a cation exchange resin,DIAION, WA30 (Mitsubishi) and an anion exchange resin, AMBERLITE IR120NA (Rohm and Haas Company). The resulting solution is concentrated withthe following apparati: EYELA Rotary Vacuum Evaporator N-N Series; EYELAWaterbath SB-450; and EYELA, Aspirator A-3 (Tokyo Rikakikai Co. LTD).The concentrated solution is crystallized at room temperature. Crystalsare optionally washed with or re-crystallized in hydrous alcohol andwater (e.g., at 4° C.) to remove the trace impurities.

[0024] Verification of Erythritol Purification

[0025] To confirm the chemical identity of the purified product, the NMRspectra of the purified product is compared to the NMR spectra of astandard, e.g., erythritol purchased from Merck Co. (NJ, USA), oranother commercial supplier. The samples are dissolved in 100% D₂O andplaced in an NMR spectrometer (Bruker AM-500, Germany). The followingconditions are used for ¹H NMR spectra: 400.135 MHz; pulse length: 4.0μs; acquisition time: 1.245 sec; pulse delay: 1 sec; chemical shifts:D₂O as 0 ppm. The following conditions are used for ¹³C NMR spectra:100.536 MHz; pulse length: 5.0 μs; acquisition time: 0.623 sec; pulsedelay: 2 sec; chemical shifts: 10 mM DSS as 0 ppm.

[0026] A skilled artisan can obtain a fungal mutant of the invention andutilize it to the fullest extent to produce erythritol based on theguidance of the following specific example, which is merelyillustrative, and not limitative of the scope of the invention. Allpublications cited herein are incorporated in their entirety byreference.

EXAMPLE

[0027] Moniliella Mutant Isolation

[0028] The erythritol-producing fungi Moniliella PTA-1230 wasmutagenized with NTG by the method described above. The procedure wasrepeated such that an improved erythritol producer isolated in one roundis used as the parent strain for the subsequent round. The N61188-12mutant strain (ATCC deposit ______) was isolated after six rounds ofmutagenesis.

[0029] The N61188-12 mutant strain and the parental PTA-1230 werecultured in broth containing 35% glucose and 1% yeast extract on rotaryshaker at 150 rpm for 6 days at the temperature of 25° C., 30° C., 34°C., and 37° C. At each of these temperatures, the glucose-to-erythritolconversion rates were respectively: 43.9%, 61.4%, 17.8%, and 2.2%, forthe N61188-12 mutant strain; and 18.9%, 30.5%, 17.9%, and 7.7% for theparental PTA-1230. At 25° C. and 30° C., the erythritol yields of theN61188-12 strain were at least twice as great as that of the PTA-1230.The 61.4% yield observed for the N61188-12 strain was unexpected, as itis remarkably close to the theoretical upper limit for completeconversion of glucose to erythritol −68%.

[0030] For the purposes of verification, pure erythritol was obtainedfrom a fermentor culture of the N61188-12 strain using theabove-described methods. The pure erythritol from N61188-12 was analyzedby nuclear magnetic resonance as described above. Its spectra wereidentical to the spectra of an erythritol standard indicating that theproduct recovered, purified, and crystallized was, indeed, erythritol.

[0031] Optimization of Erythritol Production Conditions.

[0032] The erythritol yields were determined in parallel for theparental PTA-1230 and the N61188-12 strain under conditions of varyingpH, temperature (see above), and carbon source.

[0033] pH.

[0034] The parental PTA-1230 and the N61188-12 strains were cultured in35% glucose and 1% yeast extract broth adjusted to various pH's at 30°C. on a rotary shaker at 150 rpm for 6 days. For the pH's 3.0, 4.0, 5.0,6.0, and 7.0, the erythritol yield of the PTA-1230 was 31.2%, 39.3%,38.4%, 34.4%, and 34.2% respectively, whereas the erythritol yield ofthe N61188-12 strain was 56.6%, 59.4%, 58.5%, 60.3%, and 57.3%,respectively.

[0035] Glucose concentration.

[0036] Culture broths containing 20%, 30%, 35%, 40%, and 50% glucosetogether with 1% yeast extract were prepared. Both PTA-1230 andN61188-12 strains were cultured in the above broths at 30° C. on arotary shaker at 150 rpm for 6 days. At each of these glucoseconcentrations, the erythritol yield of the PTA-1230 strain was 40.6%,37.1%, 34.5%, 29.4%, and 19.2%, respectively, whereas the erythritolyield of the N61188-12 strain was 56.3%, 57.5%, 62.8%, 55.6%, and 35.8%,respectively (Table 10). The optimal yield of the PTA-1230 strain waswith the 20% glucose solution, the yield decreasing with the increasingglucose concentration. The optimal yield of the N61188-12 strain waswith the 35% glucose broth. The yields obtained from other glucoseconcentrations, such as 20%, 30%, and 40% glucose solution, were similarto each other, while that obtained from 50% glucose solution was reducedto 35.8%. At high glucose concentrations, e.g., 40% and 50%, theerythritol yield of the N61188-12 strain was nearly twice that of thePTA-1230 strain. These results indicate the unexpectedly improvederythritol production capacity of the N61188-12 strain in comparison tothe wild type PTA-1230 strain.

[0037] Carbon Source.

[0038] The culture broths containing 35% of either glucose,maltodextrin, maltose, sucrose, fructose, or lactose as carbon source,and 1% yeast extract as nitrogen source were prepared. Both PTA-1 230and N61188-12 strains were cultured in above broths at 30° C. on arotary shaker at 150 rpm for 6 days. Respectively for glucose,maltodextrin, maltose, sucrose, fructose, or lactose, strain PTA-1230produced 120.8, 44.5, 0, 154.0, 111.0, and 0 g/L of erythritol, whereasthe N61188-12 strain produced 220.0, 15.1, 22.8, 239.4, 211.4, and 0g/L. These results indicated that sucrose has best conversion capacityfor both fungal strains, and the next being glucose, and then fructose.Notably, strain PTA-1230 cannot utilize maltose and lactose forerythritol production, whereas the N61188-12 strain can utilize maltose,but not lactose for erythritol production. For the PTA-1230 strain, theerythritol yield using sucrose as the carbon source was 27.5% higherthan that using glucose, whereas the yield was only 9% higher under thesame conditions for the N61188-12 strain.

[0039] Byproduct Accumulation.

[0040] The concentrations of the metabolic byproducts—glycerol,pentitol, and alcohol—were monitored in the aforementioned carbonsources. For example, when glucose was used as the carbon source, theconcentration of glycerol and pentitol in the PTA-1230 strain culturebroth was 36.4 and 17.2 g/L, respectively, whereas there was no glycerolpresent, and the content of pentitol was only 3.8 g/L in the N61188-12strain culture broth. Results for additional carbon sources areillustrated in Table 1.

[0041] No alcohol was producing during the fermentation of the PTA-1 230strain with glucose as the carbon source. However, in other carbonsources, both the PTA-1230 and N61188-12 strains produced alcohol forthe initial five days after inoculation. However, the alcohol wasexhausted on the 6^(th) day. The only exception was some residualalcohol (0.7 g/L) on the 6^(th) day when fructose was used for theN61188-12 culture. In sum, these results indicate that the use ofsucrose for culturing the N61188-12 strain results in a high conversioncapacity to erythritol without the accumulation of byproducts. TABLE 1Production of erythritol and byproducts of PTA-1230 and N61188-12strains Byproducts from PTA-1230 strain (g/L ) Byproducts from N61188-12strain (g/L) Carbon Source Erythritol Glycerol Pentitol AlcoholErythritol Glycerol Pentitol Alcohol glucose 120.8 36.4 17.2 0 220 0 3.80 maltodextrin 45.5 0 0 0 15.1 0 0 0 maltose 0 0 0 0 22.8 0 0 0 sucrose154 23.4 0 0 239.4 0 0 0 fructose 111 26.3 7.6 0 211.4 13.6 4.2 0.7

[0042] Gross properties of mutant strain N61188-12.

[0043] The parental PTA-1230 strain and the mutant N61188-12 strain weregrown under various conditions and compared. Their cell morphologieswere substantially the same. However, on plates, the mutant strain grewto only a quarter the size of the PTA-1230 strain. Notably, the twostrains have different physiological properties. These differences arereflected in their abilities to ferment and assimilate different sugars.The mutant N61188-12 strain can ferment galactose (Table 2), whereas thePTA-1230 strain cannot. In addition, the N61188-12 strain is unable toassimilate erythritol and mannitol (Table 3) in contrast to the PTA-1230strain. TABLE 2 Fermentation of various carbon sources Carbon sourcePTA-1230 strain N61188-12 strain glucose + + galactose − + maltose + +sucrose + + lactose − −

[0044] TABLE 3 Assimilation study of PTA-1230 and N61188-12 strains onvarious carbon sources PTA-1230 PTA-1230 Carbon source strain N61188-12strain Carbon source strain N61188-12 strain glucose + + ribitol − −galactose − − xylitol − − sorbose − − arabinitol − − glucosamine − −glucitol − − ribose − − mannitol + − xylose − − galactitol − −L-arabinose − − myo-inositol − − D-arabinose − − glucono-1,5-lactone + −rhamose − − 2-keto-gluconate − − sucrose + + gluconate − − maltose + +glucuronate − − trehalose − − galacturona − − methyl-D-glucoside − −lactate − − cellobiose + + succinate + + salicin − − citrate − −arbutin + + methanol + + melibiose − − ethanol − − lactose − − propane −− raffinose − − butane − − melezitose − − quinate − − inulin − −saccarate − − glycerol + + galactonate − − erythritol + W*

[0045] Cell Density.

[0046] Under various conditions such as temperature, pH, carbon source,and glucose concentration, the turbidity (A₆₆₀) of the culture broth forthe N61188-12 strain was less than that of the PTA-1230 strain. Overall(except for use of maltose and lactose as the carbon source), theturbidity of the culture broth for the N61188-12 strain was between 31%and 77% of that for the PTA-1230 strain. In most cases the turbidity ofthe N61188-12 strain was less than 50% of the PTA-1230 strain. Thus, itis inferred that the N61188-12 strain reduced the proportion of carbonsource applied to cell growth, and instead converted a greaterproportion of the carbon source into erythritol.

OTHER EMBODIMENTS

[0047] A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. An isolated strain of the Moniliella species,wherein the strain converts glucose to erythritol with a conversion rateof at least about 45%.
 2. The isolated strain of claim 1 wherein thestrain converts glucose to erythritol with a conversion rate of at leastabout 50%.
 3. The isolated strain of claim 2 wherein the strain convertsglucose to erythritol with a conversion rate of at least about 60%. 4.The isolated strain of claim 1 wherein the strain is a mutant of theMoniliella strain PTA-1227.
 5. The isolated strain of claim 1 whereinthe strain is a mutant of the Moniliella strain PTA-1228.
 6. Theisolated strain of claim 1 wherein the strain is a mutant of theMoniliella strain PTA-1229.
 7. The isolated strain of claim 1 whereinthe strain is a mutant of the Moniliella strain PTA-1230.
 8. Theisolated strain of claim 1 wherein the strain is a mutant of theMoniliella strain PTA-1232.
 9. The isolated strain of claim 7 whereinthe strain is N61188-12, deposited with the American Type CultureCollection with the accession number ______.
 10. A method of producingerythritol, the method comprising: growing the Moniliella strain ofclaim 1 in a culture; and purifying erythritol from the culture.
 11. Amethod of producing erythritol, the method comprising: growing theMoniliella strain of claim 2 in a culture; and purifying erythritol fromthe culture.
 12. A method of producing erythritol, the methodcomprising: growing the Moniliella strain of claim 3 in a culture; andpurifying erythritol from the culture.
 13. A method of producingerythritol, the method comprising: growing the Moniliella strain ofclaim 4 in a culture; and purifying erythritol from the culture.
 14. Amethod of producing erythritol, the method comprising: growing theMoniliella strain of claim 5 in a culture; and purifying erythritol fromthe culture.
 15. A method of producing erythritol, the methodcomprising: growing the Moniliella strain of claim 6 in a culture; andpurifying erythritol from the culture.
 16. A method of producingerythritol, the method comprising: growing the Moniliella strain ofclaim 7 in a culture; and purifying erythritol from the culture.
 17. Amethod of producing erythritol, the method comprising: growing theMoniliella strain of claim 8 in a culture; and purifying erythritol fromthe culture.
 18. A method of producing erythritol, the methodcomprising: growing the Moniliella strain of claim 9 in a culture; andpurifying erythritol from the culture.